Use of triterpensaponins, such as notoginsenoside R1 (NR1) and/or astragaloside (ASIV) for preparing medicaments

ABSTRACT

The use of triterpensaponins, like notoginsenoside R1 (NR1) and/or astragaloside (ASIV) for the production of medicaments for stimulating the fibrinolytic activity and blocking the endotoxin effect, especially for the treatment of patients and animals which suffer from endotoxin shock or to limit the endotoxin shock. Corresponding medicaments are also suitable prophylactive and therapeutic treatment of patients of coronary heart disease, peripheral arterial disease for patients who suffer from angina pectoris and for the prevention of such diseases in healthy persons.

SPECIFICATION BACKGROUND OF THE INVENTION

The fibrinolytic system serves as a basic defense mechanism to controlthe deposition of fibrin both in the vascular as well as in the extravascular systems. Proper functioning of the fibrinolytic system isnecessary on the one hand to limit haemorrhagia and on the other handthrombotic phenomena, but also to limit the formation of interstitialfibrin deposits and consequent scarring. It has been recognized that thetissue plasminogen activator (t-PA) plays an important role in theinitiation of the (extrinsic) fibrinolytic cascade by the transformationof the zymogen plasminogen into the active plasmin that degrades fibrin.It has also been found that the fibrinolytic capacity of plasma isstrongly dependent on the concentration of circulating t-PA. The t-PA inthe plasma probably stems primarily from the vascular wall where it islocalized in the endothelial cell. In addition, urokinase plasminogenactivator (u-PA) plays a role in the overall fibrinolysis. It has beenthought that this plasminogen activator--also at least partly--derivesfrom the vascular wall. The main inhibitor of fibrinolysis, plasminogenactivator inhibitor (PAI-1) is also synthesized by endothelial cells anddata exists which shows that the relative proportion between PAs andPAI-1 is important for the fibrinolytic capacity and in turn forprevention of thrombotic events like, for example, myocardialinfarction. The pharmacological regulation of t-PA, u-PA and PAI-1synthesis is therefore useful to increase insufficient endogenousfibrinolysis.

Since both t-PA and also PAI-1 are produced by endothelial cells, theregulation of their synthesis and secretion at the level of theendothelial cell provides a rapid and direct way of influencing thefibrinolytic potential of the blood. Studies recently carried out haveindicated that the production of plasminogen activators and inhibitorsin various cell types is regulated by a series of factors: The synthesisof t-PA in endothelial cells is increased by a number of stimuli such asthrombin, histamine, butyrate, retinoic acid and tumor promoters as, forexample, phorbol-12-myristate-13-acetate (PMA). Factors which regulatethe PAI-I expression in endothelial cells include lipopolysaccharides,thrombotim, interleukin-1(IL-1), tumor necrosis factor α, (TNF α),transforming growth factor β (TGF β), basic fibroblast growth factor(BFGF) and endothelial cell growth supplement in combination withheparin. None of the above-mentioned substances could, however, be usedsuccessfully in vivo.

Bacterial sepsis initiated by the over liberation of bacterialendotoxins (LPS) is a life threatening condition in which changes incoagulation and fibrinolysis initiated by LPS produce intervascularclotting and, in turn, organ failure. it is thought that LPS operatesupon endothelial cells in which the expression of tissue factor (TF) andPAI-1 are increased.

To date there is no satisfactory direct treatment of patients which cancure intervascular clotting induced by LPS and efforts to deal with thesystems triggered by LPS like, for example, hypercoagulation, are on theone hand limited to heparin and on the other by treatment of thebacterial sepsis with antibiotics. In China, the Chinese herbal drugPanax notoginseng or triterpensaponins have been used by traditionalChinese doctors to relieve pain and treat cardiovascular heart diseaseand stasis for thousands of years.

Indeed for example in L. Zechmeister, Progress in the Chemistry ofOrganic Natural Products, Vol. 46,Springer-Verlag, Vienna, N.Y., 1984,inthe chapter on "Saponins of Ginseng and Related Plants", thecharacteristics of Panax notoginseng as a tonic, a haemostat, a coronarytherapeutic and an antihaemorrhagic have been described. Also there isan indication in Chemical Abstracts 119, 85 683 as to theantihaemorrhagic effect of Panax notoginseng and in the Japanese PatentAbstracts JP Kokai No. 55-127 317,as to the antifibrinolytic effect andin JP Kokai No. 63-198 609 as to the blood flow promoting effect ofPanax notoginseng. In none of these previous references, however, is theclot dissolving effect of Panax notoginseng or the saponins isolatedtherefrom described. This clot dissolving effect of Panax notoginseng orof astragaloside has thus not heretofore been described anywhere.Generally there are hardly any therapeutically usable substances knownwhich increase endogenous fibrinolytic activity or which are effectiveagainst clot formation and the antifibrinolytic activity of endotoxins.

SUMMARY OF THE INVENTION

The use of substances to increase the fibrinolytic capacity and todirectly block the LPS effect is described here for the first time. Thesubject of this invention is the use of triterpensaponins, likenotoginsenoside R1 (NR1) and/or astragaloside (ASIV) or substances withrelated chemical structure, which differ from triterpensaponin only bytheir side chain residues and/or the glycosylation, either in the formof pure substances or as mixtures thereof, for producing medicamentswhich can be used to treat patients either parenterally or orally in theform of solutions or tablets or capsules, to increase fibrinolyticcapacity, prevent cardiovascular diseases and inhibit endotoxin effectsas, for example, in septic shock.

EXAMPLES

In all examples the following materials and methods are used.

Chemically pure notoginsenoside R1 (NR1) or chemically pureastragaloside ASIV was purchased from the National Institute for theControl of Pharmaceutical and Biological Products (Beijing, China). NR1or astragaloside ASIV are substances with the following formulae:##STR1##

NR1 or ASIV are dissolved an incubation medium and diluted to achieve afinal concentration of 0.01 to 100 μg/ml. Lipopolysaccharide(Escherechia coli lipopolysaccharide, Sero type 026:B6 prepared byphenol extraction) was obtained from Sigma (St. Louis, Mo., USA). Asolution with a concentration of 1 mg/ml in distilled water was storedat -70° C. Morpholinopropanesulfonic acid (Serva, Germany), guanidinethiocyanate (Fluka, Switzerland), piperazine-N,N'-bis(2-ethane-sulfonicacid) (PIPES; Sigma), Seakem LE agarose (FMC Bioproducts, Me., USA),dCTP Aloha-32P! (ICN Radiochemicals, Calif., USA) were obtained from theindicated firms. The remaining materials which are described in themethods have been specified in detail in the corresponding citations.

Cell Culture

Endothelial cells were isolated from fresh human umbilical cord veins bycollagenase (Sigma) by a technique similar to the protocol of Jaffe etal., J. Clin Invest 1973; 52: 2745-56. Cells from 4-6 umbilical cordswere pooled and plated in 75 cm² tissue culture flasks (Costar, Mass.,USA) which were coated with 1% calf hide gelatin (Sigma). The cells werecultivated to confluence at 37° C. in a water vapor saturated atmosphereof 95% air and 5% CO₂ in medium 199 (Sigma) to which were added 20% heatinactivated supplemented calf serum (SCS; Hyclone, Utah, USA), 100 μg/mlStreptomycin, 100 IU/ml Penicillin, 250 ng/ml Fungizon, 1 mM glutamine(JHR Biosciences, Kans., USA), 2 IU/ml Heparin (Liquemin Roche; HoffmannLa Roche, Switzerland), 50 μg/ml ECGS (Technoclone, Austria). Theendothelial character of the cells was confirmed by their typicalcobblestone morphology which was characterized by their typicalcobblestone morphology, by positive immunoflorescence with anti-VonWillebrandt Faktor VIII antibodies and by the takeup of acetylated lowdensity lipoprotein (LDL). Primary cultures at the confluence time pointwere harvested with 0.05% Trypsin/0.02% EDTA (JRH Biosciences) and wereplated in a split ratio of 1:3 in 75 cm² cell culture flasks.Subconfluence cells were allowed to grow until they attained confluenceand were harvested during the exponential cell growth with trypsin/EDTAand were frozen in 1 ml portions of medium 199 with 10% dimethylsulfoxide (DMS) in liquid nitrogen. For the experiments, the cells werethawed at 37° and were grown in 6-well plates (diameter 3.5 cm; Costar)in medium 199 to which SCS, ECGS and heparin were added in the abovegiven concentrations until reaching confluence. In all experiments cellsbetween the second and third passages were used. The cells were alwaysfed the day before the experiment with fresh medium. All of thematerials used in thee cell culture were determined to be free fromendotoxin (detection limit of the test 5 pg/ml) by the Coatest EndotoxinKit (Kabi Vitrum, Sweden).

Production of the Conditioned medium (CM) and the Extra Cellular Matrix(ECM)

Confluent cultures are washed twice with Hank's Balanced Salt solution(HBSS; Sigma) and incubated at 37° C. with 1 ml/Napf of medium 199 towhich is added 1.25% SCS and 50 μg/ml ECGS and NR1 or ASIV in theindicated concentrations. After the incubation, the cell culturesupernatant was collected and after centrifugation to remove cellfragments, was stored at -70° C. until used. The total cell count of thecorresponding cultures was determined by trypsinizing with ahemocytometer. ECM was prepared from these or similarly treated culturesaccording to the method of Mimuro et al., Blood 1987; 70: 721-28.Themonolayer was washed three times with cold phosphate buffered salinesolution (PBS:0.01 monosodium phosphate, 0.14 m NaCl, pH 7.4) and thecellular components extracted by incubation for 10 minutes at 37° C.with PBS containing 0.5% Triton X100.

The plates were washed one further time with distilled water to removeremaining cellular components and then investigated for the presence ofcell fragments microscopically. This extraction method removed visiblecell components completely from the plates and the ECM was extracted byshaving into 1 ml PBS containing 0.1% SDS after 30 minutes of incubationat 37° C. The extracts were dialyzed overnight against PBS.

Tests for t-PA antigen, uPA antigen, PAI-1 antigen, PAI-1 activity andt-PA PAI-1 complexes in CM, in the ECM and in plasma.

t-PA antigen, u-PA antigen, PAI-1 antigen and the concentrations of t-PAPAI-1 complexes were determined with specific commercially availableenzyme linked immunosorbent assays (ELISAs) (Technoclone) according tothe manufacturers instructions. The test ranges for these assays werefor t-PA between 0.3 and 2.5 ng/ml, for u-PA between 0.6 and 10 ng/ml,for PAI between 1.0 and 30 ng/ml and for t-PA PAI-1 complexes between0.2 and 20 ng/ml. The t-PA ELISA determines free t-PA and t-PA complexedwith PAI-1. The u-PA ELISA determines free u-PA and t-PA complexed withPAI-1. The PAI-1 ELISA determines free, complexed and lateral PAI-1. Thet-PA PAI-1 complex ELISA exclusively measures t-PA complexes. PAI-1activity in plasma and in CM were determined by titration assay(Technoclone) in accordance with the manufacturers instructions.

Determination of Functional Activities of tPA and PAI-1

The activities of tPA and PAI-1 was analyzed after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) utilizingfibrinautography (FA) and reverse fibrinautography (RFA). SDSpolyacrylamide gels and buffer were fabricated in accordance with theprotocol of Laemmli, Nature 1970; 227: 680-85. FA is carried out inaccordance with Granelli-Piperno et al., J. Exp Med 1978; 148: 223-34.100 μl of the corresponding samples were applied to a 10 cm longresolving gel with 10% acrylamide and a 2 cm long stacking gel with 4%acrylamide and carried out at room temperature for 16 hours or until thecolor front reached the bottom of the gel. After the electrophoresis,the gels were initially treated with 250 ml water containing 2.5% tritonX100 (serva) for 90 minutes (the Triton solution was changed after 45minutes) to neutralize the SDS and then the gel was placed on afibrin-agar-indicator film containing 1.5% agarose type L (Behring,Germany), 2 mg/ml plasminogen-rich fibrinogen (Organon Teknika, Holland)and 0.2 IU/ml bovinine thrombin. The gels were incubated at 37° C. in ahumidified chamber and at different points in time were photographed.RFA was carried out in that the gels were applied to a fibrin film whichwas basically fabricated as described above but which additionallycontained 0.4 IU/ml urokinase (Technoclone). The quantification of thetPA and PAI-1 activity in a given sample was obtained in that both lysiszones and lysis resistance zones were photographed on the indicatorfilm. These zones were outlined on transparent paper and the outlinedareas were cut out and weighed on an analytical balance. To identify theplasminogen activator contained in the CM of HUVECs, samples of the CMwere incubated at 4° C. for 24 hours with either CNBr activatedSepharose bound monoclonal anti-tPA antibody (NPW 3 VPA; Technoclone) ormonoclonal anti u-PA antibody (MPW 5 UK; Technoclone) or, as control,with Sepharose 4B (Pharmacia, Sweden). Thereafter the Sepharose wasremoved by centrifugation and 100 μl of the corresponding samples wereanalyzed by the above-described SDS-PAGE and subsequent FA.

Preparation of the cell lysate and measurement of the TF activity

HUVECs were incubated for 6 hours at 37° C. in medium 199 with LPSand/or NRI. The cells were washed 3 times with clotting buffer (130 mMNaCl, 8 mM Na-barbital and 12 mM Na-acetate, pH=7.4) and taken up in 300μl clotting buffer by scraping. The scraped off cells were frozen andthawed 3 times. The cell lysate was checked for TF activity in a onestep clotting assay. 100 μl of the cell lysate was incubated with 100 μl20 mM CaCl₂ at 37° C. for 5 minutes in preheated plastic tubes in acoagulometer (H. Amelumg GmbH, Germany). The clotting was initiated by100 μl preheated normal human citrate plasma or by the addition ofFactor X deficient plasma (SIGMA). The TF activity was quantified with astandard curve (LOG-LOG PLOT) constructed with rabbit brainthromboplastin (SIGMA). 100 mU activity is defined as that whichprovides a coagulation time of 20 seconds in a standard test with normalhuman plasma. The observed coagulator activities corresponded to the TFactivity because no procoagulant activity of the endothelial cells wasdetected when Factor X deficient plasma was used instead of normalplasma.

Quantification of the t-PA, PAI-1,TF mRNA quantities by Northern BlotAnalysis.

The total cellular RNA is isolated from endothelial cells by acidquandine thiocyanate-phenol chloroform extraction as described byChomzynski und Sacchi, Anal Biochem 1987; 162: 156-9. The RNAprecipitate was resuspended in 50 μl 0.5% SDS and the concentrationfixed at 260 nm. For the Northern Blot Analysis the RNA samples weresubjected to electrophorese in 1.2% agarose gel and the fractionated DNAwas transferred to a Duralon-UV ™ membrane (Stratagene, Calif., USA) bythe capillary effect. The RNA blots were introduced into seal-a-mealbags and prehybridized in 50 mM PIPES, 100 mM NaCl, 50 mM Na phosphate,1 mM EDTA containing 5% SDS for at least 3 hours at 57° C. Theprehybridization buffer was discarded and replaced by freshprehyberization buffer with 10⁶ cpm/ml of ³² P marked cDNA probe foreither human t-PA, human PA1, human TF or rat glycerylaldehyde-3-phosphate dehydrogenase which is used as an internalstandard. The CDNA fragments were radioactively marked with a RandomPrime DNA labelling kit (Boehringer Manneheim, Germany).

Animal Experiments

In these studies exclusively male Balb C Mice (18-30 gm body weight)were used. All experiments were carried out under ether anesthesia. Themice were injected intravenously via the tail vein with LPS (10 ng/g)and/or NR1 (1 μg/g) in a volume of 5 μg/l. At the times specified, bloodspecimens were obtained and anti-coagulated with sodium citrate (0.13Mfinal concentration). Thrombocyte-free plasma was prepared bycentrifugation at 2500 g for 30 minutes at 4° C. and stored at -70° C.until tested.

Dispensing of NR1 Containing Extracts to Humans

A notoginseng R1 (NR1) containing extract was administered to sixhealthy volunteers in a time interval of 24 hours 4×100 mg-equivalent.Blood was taken directly prior to the first administration and directlyafter the last administration. In these blood samples, corresponding tothe above given methods, the tissue plasminogen activator inhibitor I(PAI-1) antigen, tissue plasminogen antigen (tPA) and urokinaseplasminogen activator (u-PA) antigen were determined.

Statistical Analysis

The results are reported as mean ± standard deviation. An unpairedStudent's t-test was used to determine significance.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A is a graph showing the effect of Notoginsenoside R1concentration in μg/ml on Tissue Plasminogen Activator (tPA) Antigen inng/10⁵ human umbilical vein endothelium cells (HUVEC).

FIG. 1B is a graph showing the effect of Notoginsenoside R1concentration in μg/ml on Tissue Plasminogen Activator-PlasminogenActivator Inhibitor (t-PA-PAI-1) Complex in ng/10⁵ HUVECS.

FIG. 1C is a graph showing the effect of Notoginsenoside R1concentration in μg/ml on Plasminogen Activator Inhibitor (PAI-1) inμg/10⁵ HUVECs.

FIG. 2 is a graph showing the effect of Notoginsenoside R1 concentrationin μg/ml on PAI-1 concentration in μg/10⁵ HUVECs.

FIG. 3a is a set of two graphs plotting hours after administeringNotogenoside R1 (NR1) or an inert control to HUVECs versus t-PA AntigenConcentration in (ng/10⁵ HUVECS). The top graph shows the t-PA Antigenconcentration after administration of NR1 and the bottom graph shows thet-PA Antigen Concentration after administration of the control.

FIG. 3b is a set of two graphs plotting hours after administeringNotogenoside R1 (NR1) or an inert control to HUVECs versus PAI-1 AntigenConcentration in (ng/10⁵ HUVECs). The top graph shows the PAI-1 AntigenConcentration after administration of NR1 and the bottom graph shows thePAI-1 Antigen Concentration after administration of the control.

FIG. 4 is a series of six fibrinautography runs on HUVECs cell matrix(CM) samples.

FIG. 5A is a combination of two graph and fibrinautography (FA) datawhere concentration of NR1 is plotted against relative units of asurface region.

FIG. 5B is a combination of a graph and reverse fibrinautography (RFA)data where concentration of NR1 is plotted against relative units of asurface region.

FIG. 6 includes a set of three autoradiograms obtained by Northern BlotAnalysis of RNA extracts for untreated and NR1 treated HUVECs using ³² Pmarked CDNA probes for tPA, PAI-1 and GAPDH mRNA. Also included is a bargraph interpreting the Northern Blot data concerning the effect of NR1on HUVEC expression of tPA and PAI-1.

FIG. 7 is a bar graph illustrating the up-regulation of PAI-1 antigenmeasured after exposure of HUVECs to bacterial endotoxins (LPS) (1μg/ml) for 12 hours antagonized by simultaneous treatment with variousconcentrations of NR1.

FIG. 8 is an autoradiogram obtained by Northern Blot Analysis of RNAextracts treated with LPS, NR1 and both LPS and NR1 to show the effectsof LPS, NR1, and LPS and NR1 on PAI-1 activity. Also included is a bargraph interpreting the data concerning the effect on PAI-1 activity.

FIG. 9 is a series of line graphs plotting time against PAI-1 Activityin mice who were injected with LPS, NR1, LPS and NR1 or an inertcontrol.

FIG. 10 is a series of bar graphs showing Tissue Factor (TF) Activityafter administration to HUVECs of 1 μg/ml LPS alone and with variousconcentrations of NR1.

FIG. 11 is a set of two autoradiograms obtained by Northern BlotAnalysis measuring TF mRNA and GAPDH mRNA. FIG. 11 also includes a bargraph showing the effect on TF mRNA of NR1, LPS and NR1 and LPS.

FIG. 12 is a graph plotting the concentration of astragaloside (ASIV)against tPA activity.

FIG. 13 is a graph plotting the concentration of astragaloside (ASIV)against PAI-1 Antigen activity.

FIG. 14 is a series of three autoradiograms obtained by Northern BlotAnalysis measuring tPA, PAI-1 and GAPDH mRNA. FIG. 14 also includes abar graph showing the effect of administering ASIV to HUVECs over aperiod of time on the mRNA of tPA and PAI-1.

FIG. 15 is a series of bar graphs showing the effect of treating HUVECswith LPS and/or ASIV on PAI-1 antigen production.

FIG. 16 is a graph plotting time against TF Activity in HUVECs aftertreatment with LPS and/or ASIV.

FIG. 17 is a series of two autoradiograms obtained by Northern BlotAnalysis measuring TF and GAPDH mRNA. FIG. 17 also includes a bar graphshowing the effect of administering ASIV, LPS and ASIV on TF MRNA inHUVECs.

FIG. 18 is a set of bar graphs showing the effects of extractscontaining NR1 on fibrinolytic activity (tPA activity) in humans againstan untreated control.

FIG. 19 is a set of bar graphs showing the effects of extractscontaining NR1 on fibrinolytic activity (uPA activity) in humans againstan untreated control.

FIG. 20 is a set of bar graphs showing the effects of extractscontaining NR1 on fibrinolytic activity (PAI-1 activity) in humansagainst an untreated control.

EXAMPLE 1

Effect of NR1 on the production of tPA, u-PA and PAI-I-cultivated humanumbilical vein endothelium cells.

Effect of notoginsenoside R1 (NR1) on production of tPA antigen (FieldA), tPA PAI-1 complex (Field B) and PAI-1 antigen (Field C) in culturedHUVECs (FIG. 1).

HUVECs were incubated for 24 hours at different concentrations of NR1(0.01 to 100 μg/ml) and the CM was analyzed for tPA antigen PAI-1antigen and tPA PAI-1 complexes using the materials and methodsdescribed. The results are the average values of three experiments ascarried out in triplicate. The values are given as means ± S.D. inFIG. 1. Significances are given by comparison with the controls(*p<0.05: **p<0.01: ***p<0.001) as indicated in FIG. 1a the treatment ofHUVECs with increasing doses of NR1 for 24 hours gives rise to a dosedependent increase in the tPA antigens in the CM of such treated cells:maximum effects were reached with 100 μg/ml NR1 (100 mg/ml NR1: 9.6±0.7ng/10⁵ cells/24 hour; controls: 5.8±0.4 ng/10⁵ /24 hours; n=9,p<0.05).As has been indicated in FIG. 1B, the tPA PAI-1 complexes in CM increasein a similar way in the presence of increasing concentrations of NR1(100 μg/ml NR1: 63.5±2.6 ng/10⁵ cells/24 hours; controls:40.2±7 ng/10⁵cells/24 hours/n=9 p<0.01).

Effect of notoginsenoside (NR1) on the PAI-1 antigen production in theECM of cultured HUVECs (FIG. 2)

HUVECs are incubated for 24 hours with different concentrations of NR1(0.01-100 μg/ml). The ECM was investigated using the materials andmethods described, after recovery and on PAI-1 antigen. The values aremeans ±SD from 6 independent wells. PAI-1 antigen in CM and in the ECMof NR1 treated HUVECs not significantly altered by comparison to thecontrols (Cm: 100 μg NR1/ml: 2.92±0.32 μg/10⁵ cells/24 hours; control:2.78±0.45 μg/10⁵ cells/24 hours; n=9. ECM=100 μg.ml NR1:4255±3.15ng/ml/24 hours; control: 42.27±1.66 ng/ml/n=6). (FIG. 1C, FIG. 2).

Time course of tPA antigen (Field A) and PAI-1 Antigen (Field B)Production of Cultured HUVECs After Treatment with Notoginsenoside (NR1)(FIG. 3)

HUVECs are incubated for the indicated time periods in the absence (opencircles) or presence of 100 μg/ml NR1 (full circles). At the indicatedtimes, the corresponding CM was harvested and using the materials andmethods described were tested for tPA antigen and PAI-1 antigen. Theresults are given in terms of the means of 3 experiments as carried outin triplicate. The values are given as mean values ±S.D., *p<0.05;**p<0.01. As shown in FIG. 3 the tPA antigen increases in CM with a timedeficiency on HUVECs which are treated for 6, 12 or 24 hours with 100μg/ml NR1 in comparison to the control. Whereas the PAI-1 antigen in theCM of cells treated in this manner was not significantly changed.

The notoginsenoside R1 affects the u-PA antigen secretion of culturedHUVECs. When the CM harvested from HUVECs which were incubated in thepresence of 100 μg/ml NR1 on u-PA antigen, a significant change is seenin the amount of the u-PA antigen product in these cells by comparisonto cm from HUVECs which is cultured under control conditions (100 μg/mlNR1: 2.9±0.6 ng/10⁶ cells per 24 hours).

EXAMPLE 2

Effect of NR1 on the tPA and PAI-1 Activity in Cultured Human UmbilicalVein Endothelial Cells

Notoginsenoside R1 increases tPA activity and reduces the PAI-1 activityof cultured HUVECs.

Fibrinautography (FA) of HUVECs CM samples: (FIG. 4)

CM from HUVECs is collected after 24 hours and is harvested with SDSPAGE and subsequent FA as described under materials and methods. Lane 1:untreated CM from HUVECs; Lane 2: CM from HUVECs which has beenpreincubated on Sepharose coupled monoclonal anti-t-pA antibodies; Lane3 CM from HUVECs which has been preincubated with Sepharose coupledmonoclonyl anti u-PA antibodies; Lane 4: cm from HUVECs that has beenpreincubated with Sepharose 4B; Lane 5: purified human tPA; Lane 6:purified human uPA.

When CM recovered from HUVECs which have been incubated for 24 hoursunder control conditions with SDS-PAGE and are analyzed by FA, it isfound that there are two dominant lysis zones with an apparent molecularweight of 70,000 and 120,000. That lysis zones could be depleted bypreincubation with monoclonal anti-t-PA antibodies but could not beremoved by preincubation with monoclonal anti u-PA antibodies (FIG. 4).Therefore, it has been concluded that the lysis zone at 70 kDa wascaused by free t-PA and the high molecular lysis zone stemmed from t-PAcomplexed with PAI-1.

Effect of Notoginsenoside R1 (NR1) on the tPA activity and on theactivity associated with the tPA PAI-1 complex (Field A) and on thePAI-1 activity (Field B) in cultured HUVECs after analysis withfibrinautography (FA) and reverse fibrinautography (RFA) (FIG. 5).

After the incubation of confluent HUVECs for 24 hours with differentconcentrations of (NR1) (0.001-100 μg/ml), the cm was subjected toSDS-PAGE and subsequent FA or RFA as described under materials andmethods. The lysis zones and the lysis resistance zones in this FIG.were transferred to transparent paper, cut out and weighed on ananalytical balance. The weight of this transparent paper was plottedagainst the concentration of (NR1) (lower field). The data representresults of one of three separate experiments which indicate similarresults. Lane 1: Control; Lane 2: 0.01 μg/ml NR1; Lane 3: 0.1 μg/ml NR1;Lane 4: 1.0 μg/ml NR1; Lane 5: 10 μg/ml NR1; Lane 100 μg/ml NR1. Whenthe CM from HUVECs is cultured with or without increasing concentrationsof NR1 for 24 hours, and was analyzed with FA or RFA, a dose dependentincrease in the size of the lysis zones is detected while the sizes ofthe lysis resistant zones decrease with the increasing amounts of NR1(FIG. 5A and B). When the size of the lysis zones or the lysisresistance zones is quantified as described under materials and methodsan up to 3-fold increase in the tPA-dependent lysis is established incontrast to the PAI-1 dependent lysis resistance which is reduced to 20%in the presence of 100 μg/ml NR1 as compared to control (FIG. 5A and B).

EXAMPLE 3

Effect of NR1 on the t-PA and PAI-1 mRNA-Cultured Human Umbilical VeinEndothelial Cells

Effect of notoginsenoside R1 (NR1) upon tPA and PAI-1 mNRA expression inHUVECs (FIG. 6).

Confluent HUVECs were incubated for 12 hours in the absence or in thepresence of NR1 (100 μg/ml). The Northern Blot Analysis of the RNAextracts for untreated and NR1 treated HUVECs was effected using ³² pmarked cDNA probes for tPA, PAI-1 and GAPDH mRNA. The intensities of thebands of the autoradio diagram were evaluated with the aid ofdensitometry and the specific mRNA was normalized against GAPDH mRNA forthe specific mRNA for tPA or PAI-1 to take into account differences inthe loading. The signal intensities were compared as the ratio of thesignals with NR1 treated HUVECs by comparison to signals from untreatedcontrol cells. These data include the results of two independentexperiments which showed similar results. As illustrated in FIG. 6, thestimulating effect of NR1 on the secretion of tPA in HUVECs is alsoreflected in the level of the specific mRNA expression. The tPA specificmRNA increases up to a two-fold value in 12 hours with 100 μg/ml NR1treated HUVECs whereas the PAI-1 specific mNRA expression was notcontrolled by NR1 (3.2 kb:82% of the control, 2.2 kb:86% of thecontrol). When North Blot experiments were carried out in the presenceof 10 μg/ml cyclohexamide, the stimulating effect of NR1 on the tPAspecific mRNA was inhibited (data not shown).

EXAMPLE 4

Effects of NR1 on the up-regulation of PAI-1 antigen, activity and PAI-1MRNA in vitro by endotoxin.

As illustrated in FIG. 7, the up-regulation of PAI-1 antigen measuredafter exposure of cells to LPS (1 μg/ml) for 12 hours is antagonized bysimultaneous treatment with various concentrations of NR1. The extent ofthe antagonism was dose dependent upon NR1 concentration (0.1-100 μg/ml)and the LPS induced increase in the PAI-1 antigen was significantlyreduced by the co-incubation of the cells with 100 μg/ml NR1 (controlcells: 347±34 ng/10⁵ cells/12 hours, LPS treated cells: 946±42 ng/10⁵cells/12 hours, LPS and NR1 treated cells: 469±29 ng/10⁵ cells/12hours). The change in PAI-1 activity of the cells was parallel to thechange in PAI-1 antigen (control cells: 5.48±0.8 μ/10⁵ cells 12 hours,LPS treated cells: 8.22±0.18 μ/10⁵ cells/12 hours, LPS and NR1 treatedcells: 4.77±0.26 μ/10⁵ cells/12 hours/n=6). The mRNA for PAI-1 wasmeasured in cells which were treated with one μg/ml LPS and/or 100 μg/mlNR1. The increase induced by LPS to a two-fold amount of PAI-1 specificmRNA (3.2 kb) was reduced in the presence of both LPS and NR1 to a 1.37fold increase (FIG. 8).

EXAMPLE 5

Effect of NR1 on the LPS induced upregulation of the PAI-1 activity invivo.

In vivo studies indicate that the injection of LPS in mice results in arapid increase in the plasma PAI-1 activity. With an LPS dose of 10 ng/g(body weight) an increase from significant to 7 fold above the controlvalue can be reached two hours after the injection while maximum valueis reached 4 hours after injection. At a later time, the PAI-1 activityreturned gradually to normal values. By contrast, the PAI-1 activity inanimals treated simultaneously with LPS and NR1 (1 μg/g) returned to thecontrol value 4 hours after the injection (LPS treated group: 11:3±3.1U/ml, LPS and NR1 treated group: 4.3±1.0 U/ml, control group: 4.9±0.3U/ml, n=5-8) (FIG. 9).

EXAMPLE 6

Effects of NR1 on the endotoxin (LPS) induced TF activity and mRNA incultured HUVECs:

In untreated HUVECs only a very small amount of TF activity is found(0.78±0.15 mU/10⁶ cells, n=9). The TF activity increases in HUVECs aftertreatment with LPS (1 μg/ml for 6 hours) to a value of 88.6±6.5 mU/10⁶cells (n=6). The TF activity measured in HUVEC after 6 hours oftreatment with 1 μg/ml LPS is significantly antagonized by coincubationwith NR1 (LPS and NR1 treated cells: 56.0±1.9 mU/10⁶ cells). The extentof the antagonism was also dose dependent with respect to NR1concentration, whereby with 100 μg/ml MR1 an approximately 36.8 %inhibition was achieved (FIG. 10) a significant increase in the TF mRNAwas observed after treatment of HUVEC with LPS which reached a 9 foldincrease (2.4±3.1±3.5 kb) over control values after 2 hours. The TF mRNAvalues increased by LPS were antagonized in a similar way bycoincubation with RN1;the TF mRNA was reduced to a 4-fold value over thecontrol. The treatment of the cells with 100 μg/ml NR1 alone reduced theTF mRNA values to 40% of the control values (FIG. 11).

EXAMPLE 7

Effect of astragaloside (ASIV) on the antigen concentrations of tPA andPAI in human endothelial cell culture.

Effect of astragaloside IV (ASIV) on tPA antigen and PAI-1 antigenproduction in culture HUVECs (FIGS. 12 and 13).

HUVECs were incubated for 24 hours with different concentrations of ASIV(0.01-100 μg/ml) and the CM was analyzed for tPA antigen and PAI-1antigen as described under materials and methods. The results are themean values of three experiments, each in triplicate. The values are themean values ±S.D. indicated in FIGS. 12 and 13. Significances are incomparison to the control (* p<0.05; ** p<0.01; *** p<0.001). Asindicated in FIG. 12, the treatment of HUVECs with increasingconcentration of ASIV for 24 hours results in a dose dependent increaseof the tPA antigens in the CM of such treated cells: maximum effect isreached at 100 μg/ml ASIV.

FIG. 13 shows the effect on the PAI-1 antigen production in the CM ofcultured HUVECs (FIG. 13). HUVECs were incubated for 24 hours withdifferent concentrations of ASIV (0.01-100 μg/ml). The CM was recoveredas described under materials and methods and investigated for PAI-1antigen. The values are mean values ±S.D. from three investigationscarried out each in triplicate. Antigen in CM was significantly alteredwith ASIV treated HUVECs in comparison to the controls.

EXAMPLE 8

Effect of astragaloside on messenger ribo nucleic acid (mRNA) of tPA andPAI-1 in human endothelial cells.

Effect of astragaloside ASIV upon the tPA and PAI-1 mRNA expression inHUVECs: (FIG. 14)

Confluent HUVECs were incubated for 6, 12 and 24 hours in the absence orpresence of ASIV (100 μg/ml) the Northern Blot Analysis of the RNAextracts from untreated and ASIV treated HUVECs was carried out with theuse of 32 P marked CDNA probes for tPA, PAI-1 and GAPDH mRNA. Theintensity of the bands of the autoradiogram were evaluated bydensitometry and the specific mRNA for tPA or PAI-1 were normalizedagainst GAPDH mRNA to determine differences in the loading. The signalintensities were compared as the ratios of the signals with NR1 treatedHUVECs in comparison to signals from untreated control cells. These datawere indicated as results of two independent experiments with similarresults. As shown in FIG. 14, the stimulating effect of ASIV on thesecretion of tPA and on the inhibiting effect of the secretion of PAI-1in HUVECs is also reflected in the level of the specific mRNAexpression.

EXAMPLE 9

Effect of astragaloside A IV on the PAI-1 and tissue factor expressionin endotoxin treated endothelial cell cultures.

As indicated in FIG. 15, the upregulation of the PAI-1 antigen aftertreatment of the cells with LPS in different concentrations (0.1-10μg/ml for 12 hours) by simultaneous treatment with ASIV is significantlyreduced (** p<0.01; ***p<0.001). The extent of the antagonism was dosedependent on the ASIV concentration (0.1-100 μg/ml). The change in thePAI-1 activity of the cells was parallel to the change in PAI-1 antigen(data not shown). FIG. 16 shows the effect of ASIV on the endotoxininduced expression of tissue factor (TF) in HUMECs. ASIV antagonizescompletely the LPS induced TF upregulation.

A significant increase in the TF mRNA is observed after treatment ofHUVECs with LPS. The LPS increased TF mRNA value was antagonized in asimilar manner by coincubation with ASIV (FIG. 17).

EXAMPLE 10

Effects of extracts which contain notoginseng R1 on the fibrinolyticactivity in humans.

In FIGS. 18, 19 and 20 the effects of NR1 containing extracts on tPA,PAI and uPA on healthy test subjects is given. There is an increase oftPA and a decrease in PAI-1 which correspond to the effects of NR1 orASIV in tissue culture.

We claim:
 1. A method of directly blocking liberation of a bacterialendotoxin in a mammalian subject by administering a therapeuticallyeffective amount of a triterpensaponin to the subject in need oftreatment.
 2. The method of directly blocking liberation of a bacterialendotoxin defined in claim 1 wherein the triterpensaponin isnotoginsenoside or astragaloside.
 3. The method of directly blockingliberation of a bacterial endotoxin defined in claim 1 wherein thetriterpensaponin is derived from Panax notoginseng.
 4. A method forregulating synthesis of tissue plasminogen activator and plasminogenactivator inhibitor-1 in endothelial cells of a mammalian subject inneed of said treatment which comprises the step of administering atherapeutically effective amount of a triterpensaponin selected from thegroup consisting of a notoginsenoside or an astragaloside to themammalian subject.
 5. The method for regulating synthesis of tissueplasminogen activator and plasminogen activator inhibitor-1 defined inclaim 4 wherein the triterpensaponin is Notoginsenoside R1 orAstragaloside IV.